JP2012083193A - Method for detecting glycosylation of histone protein, and antibody to be used for the same - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method enabling glycosylation of histone protein to be detected and an antibody to be used for the method.SOLUTION: It is found that histones (histone H2B, histone H2A, histone H3, histone H4) are glycosylated (O-GlcNAcylated) in a nucleus, more particularly, in histone H2B, sites to be GlcNAcylated are the 91st serine residue, the 112th serine residue, and the 123rd serine residue. Additionally, an antibody which binds to a glycosylated site of histone H2B was made and further analyses were performed. Consequently, it is also found that the glycosylation depends on extracellular glucose concentration via HBP, and the enhancement of the glycosylation induces the ubiquitination of histone H2B and the methylation of histone H3, as well as activates the transcription of PGK1 genes or the like.

Description

  The present invention relates to a method for detecting glycosylation of a histone protein, and an antibody used in the method. More specifically, the present invention relates to a method for detecting glycosylation of a histone protein, a method for evaluating serum glucose level using glycosylation of histone protein as an index, and a method for examining metabolic syndrome And a method for screening drug candidate compounds for metabolic syndrome. Furthermore, the present invention relates to an antibody that binds to a glycosylated site of histone protein, an agent for detecting glycosylation of histone protein, and an agent for evaluating serum glucose level, which comprises said antibody as an active ingredient. And a drug for examining metabolic syndrome.

  It is known that the chromatin structure is controlled by various modifications in histones, as well as rearrangement to promote transcription, ie, methylation of DNA using a number of non-histone proteins (Non-Patent Documents 1 to 4). 5). In such chemical modifications, post-translational modifications of histones have emerged as important determinants for chromatin reorganization, and the specific combination of reversible histone modifications is commonly referred to as the “histone code”. It is also known that it affects the active state of chromatin (Non-patent Document 6). Although this specific combination occurs epigenetically and is stable, some of these are temporarily reconstituted in response to extracellular state and cellular activity (Non-Patent Documents). 7-8). At the present stage, the effect of eight types of modification in chromatin rearrangement has been proven (Non-Patent Document 6). Among them, histone acetylation, methylation and ubiquitination at specific residues at the histone end are associated with gene expression control (Non-patent Documents 9 to 10). That is, the induction of gene expression in euchromatinization generally involves the fourth lysine residue (H3K4) and 36th lysine residue (H3K36) of histone H3 associated with hyperacetylation of histone H3. At the same time as methylation in In contrast, methylation of H3K9 and H3K27 triggers chromatin inactivation (heterochromatinization) following histone deacetylation. Dynamic and reversible histone methylation and acetylation in accordance with the initiation of transcription is also important in the transcription elongation process and the transcription termination process (Non-patent Document 8). Furthermore, in addition to these modifications, it has recently been proved that monoubiquitination (H2B-K120-ub) at the 120th lysine residue of histone H2B is essential for the initiation process of post-transcriptional modification (Non-patent Document 11). ). Consistent with the importance of histone modifications in such transcriptional regulation, both functionally opposed enzymes that introduce / remove histone modifications function as co-regulators for DNA-binding transcription factors. It is known to do (Non-Patent Documents 12 to 14).

  However, the understanding of histone modifications related to basic transcription at the molecular level is not sufficient, and in particular, the molecular relationship between H2B-K120-ub and other histone modifications has not been clarified.

  On the other hand, it is known that the O-linked N-acetylglucosamine (GlcNAc) conversion of a protein is a reversible phenomenon as in the case of histone modification as described above (Non-Patent Documents 15 to 16). The modification reaction is catalyzed by two enzymes, O-linked N-acetylglucosaminyltransferase (OGT) and O-linked N-acetylglucosaminase (OGA), which are modified in both cytoplasmic and nucleoproteins. Has been detected. The N-acetylglucosamine moiety is derived from UDP-GlcNAc produced in the cytosol by the hexosamine biosynthetic pathway (HBP). Since the initial source of UDP-GlcNAc is extracellular glucose, it has also been shown that the degree of O-linked N-acetylglucosamination of proteins varies with extracellular glucose concentration and intracellular HBP activity. .

  However, unlike N-linked polysaccharide chains that are bound to proteins, little is known about the physiological role of O-linked N-acetylglucosamine moieties. In particular, the importance of monoglycosylation has been suggested as related to energy metabolism (and its abnormalities) (Non-patent Documents 17 to 18), but it remained unresolved because there was no precise detection system. . In particular, mass spectrometric measurement of O-linked N-acetylglucosamine of proteins has been hindered because the structure is destroyed during the ionization process (Non-patent Document 19). Furthermore, this modification has a low molecular weight and a neutral charge, and is difficult to detect because it results in a small shift in gel electrophoresis. Therefore, there is no analytical method for precisely evaluating this reversible modification other than immunodetection using specific antibodies (Non-Patent Documents 20 to 21). However, since the specific recognition of the antibody is affected by the three-dimensional structure of the bound protein, the antibody could not be used corresponding to all O-linked N-acetylglucosamined proteins. Nonetheless, with the recent improvements in mass spectrometry approaches, the functional characterization of monoglycosylated proteins involved in key intracellular events has led to the physiological nature of O-linked N-acetylglucosamined proteins. The importance is shown. Furthermore, the present inventor has found that MLL5, an H3K4 methyltransferase, is a substrate for OGT, whereby O-linked N-acetylglucosamination of nucleoproteins is an important modification for life support in chromatin reorganization. (Non-Patent Documents 22 to 23).

  Thus, although it has been demonstrated that OGT contributes to transcriptional regulation through sugar modification of histone modifying enzymes, the direct relationship between histone and sugar modification (glycosylation) has not yet been clarified. It wasn't. In addition, since O-linked N-acetylglucosamine is dependent on extracellular glucose concentration, the relationship between the sugar modification reaction in the nucleus and metabolic diseases (metabolic syndrome) including diabetes is still clear. It wasn't.

Rosenfeld et al., Genes Dev, 2006, 20, 11 (1405) Li et al., Cell, 2007, 128, 4, 707 Strahl et al., Nature, 2000, 403, 6765, page 41. Zhang et al., Genes Dev, 2001, 15, 18, 2343 Kouzarides et al., Cell, 2007, 128, 4, 693 Campos et al., Annu Rev Genet, 2009, 43, 559 Bhaumik et al., Nat Struct Mol Biol, 2007, 14, 11, 1008 Shi et al., Nat Rev Genet, 2007, 8, 11, 820 Berger, et al., Nature, 2007, 447, 7143, page 407. Weake et al., Mol Cell, 2008, 29, 6, 653 Kim, et al., Cell, 2009, Vol. 137, No. 3, p. 459 Fujiki et al., EMBO J, 2005, 24, 22, 3881 Yokoyama et al., Genes Cells, August 2010, 15, No. 8, page 867. Sawatsubashi et al., Genes Dev, 2009, 24, 2, 159 Hart et al., Nature, 2007, 446, 7139, page 1017 Love et al., Sci STKE, November 2005, 2005, No. 312, page review13 Dentin et al., Science, 2008, 319, 5868, 1402 Yang et al., Nature, 2008, 451, 7181, page 964. Chalkley et al., Proc Natl Acad Sci USA, 2009, 106, 22, 8894. Khidekel et al., Nat Chem Biol, 2007, 3, 6, 339 Teo et al., Nat Chem Biol, 2010, 6, 5, 338 Fujiki et al., Nature, 2009, 459, 7245, page 455. Chikanishi et al., Biochem Biophys Res Commun, 2010, 394, 4, 865

  The present invention has been made in view of the above-described problems of the prior art, and its purpose is to clarify the direct relationship between histone proteins and sugar modifications, thereby detecting sugar modifications in histone proteins. It is to provide a method that makes it possible. A further object of the present invention is to provide an antibody capable of detecting a sugar modification of a histone protein.

  As a result of intensive studies to achieve the above object, the present inventors have glycosylated histones (histone H2B, histone H2A, histone H3, histone H4) in the nucleus (O-linked N-acetylglucosamine). In particular, in histone H2B, it has been found that the sites to be N-acetylglucosamined are the 91st serine residue, the 112th serine residue, and the 123rd serine residue. Furthermore, the present inventors made an antibody that binds to the glycosylated site of histone H2B, and further studied the mechanism of the glycosylation. As a result, the glycosylation was caused by HBP-mediated extracellular glucose concentration. It is also found that this enhanced glycosylation induces ubiquitination of histone H2B and methylation of histone H3, and thus activates transcription of the PGK1 gene and the like, thereby completing the present invention. It came.

More specifically, the present invention provides the following inventions.
(1) A method for detecting glycosylation of a histone protein comprising the following steps (a) to (b): (a) a step of preparing a sample containing a histone protein (b) Detecting a histone protein.
(2) A method for evaluating serum glucose level, comprising detecting a glycosylated histone protein, comprising the following steps (a) to (b): (a) a sample containing histone protein (B) detecting glycosylated histone protein in the sample.
(3) A method for examining metabolic syndrome, characterized by detecting glycosylated histone protein, comprising the following steps (a) to (b): (a) preparing a sample containing histone protein (B) detecting a glycosylated histone protein in the sample.
(4) A method for screening a drug candidate compound against metabolic syndrome, comprising the following steps (a) to (d): (a) a step of preparing a sample containing histone protein (b) a test compound in the sample (C) the step of detecting glycosylated histone protein in the sample contacted with the test compound (d) the glycosylated detected in (c) as compared with the case where the test compound is not contacted Selecting a compound that reduces the amount of histone protein.
(5) The method according to any one of (1) to (4), wherein the detection uses an antibody that binds to a glycosylated site of the histone protein.
(6) The method according to any one of (1) to (5), wherein the glycosylation is N-acetylglucosamine.
(7) The method according to any one of (1) to (6), wherein the histone protein is at least one protein selected from the group consisting of histone H2A and histone H2B.
(8) The histone protein is histone H2B, and the site is at least one serine residue selected from the group consisting of the 91st serine residue, the 112th serine residue, and the 123rd serine residue The method according to any one of (1) to (7).
(9) In any one of (1) to (8), the glycosylation is N-acetylglucosamine, the histone protein is histone H2B, and the site is the 112th serine residue. The method described.
(10) An antibody that binds to a glycosylated site of a histone protein.
(11) The antibody according to (10), wherein the glycosylation is N-acetylglucosamine.
(12) The antibody according to (10) or (11), wherein the histone protein is at least one protein selected from the group consisting of histone H2A and histone H2B.
(13) The histone protein is histone H2B, and the site is at least one serine residue selected from the group consisting of a 91st serine residue, a 112th serine residue, and a 123rd serine residue The antibody according to any one of (10) to (12).
(14) In any one of (10) to (13), the histone protein is histone H2B, the site is the 112th serine residue, and the glycosylation is N-acetylglucosamination. The antibody described.
(15) A drug for detecting glycosylation of a histone core, comprising the antibody according to any one of (10) to (14) as an active ingredient.
(16) A drug for evaluating serum glucose level, comprising the antibody according to any one of (10) to (14) as an active ingredient.
(17) A drug for examining metabolic syndrome, comprising the antibody according to any one of (10) to (14) as an active ingredient.

  In the present invention, the histone protein is glycosylated and the site where the glycosylation is performed has been identified. As a result, glycosylation of histone proteins can be detected, and as a result, it becomes possible to examine diseases related to the glycosylation and develop therapeutic drugs. In addition, the provision of the antibody of the present invention makes it possible to easily detect the glycosylation and examine the disease.

Purification scheme of N-acetylglucosamined glycoprotein from Hela S3 cell-derived chromatin and O-linked type in HeLa S3 cells cultured in DMEM containing 1 g / L (Low) glucose and DMEM containing 4.5 g / L (High) glucose It is the schematic which shows the result of having analyzed the amount of N-acetylglucosamine by Western blotting (WB). It is the photograph of the gel which gave the silver stain which shows the result of the refinement | purification using a WGA lectin and (alpha) -O-linked N-acetylglucosamine ((alpha) -GlcNAc, RL2) antibody. In the figure, “In” indicates a chromatin extract, “E” of WGA indicates an eluted fraction from the WGA lectin agarose column, and “R” of WGA indicates a residual fraction of the WGA lectin agarose column. In addition, IgG1 is a negative control for α-GlcNAc, and # 1, # 2, and # 3 in IgG1 and α-GlcNAc are the first time when purified with each antibody (eluted with GlcNAc-O-Ser). ) The elution fractions for the second time (eluted with GlcNAc-O-Ser) and the third time (eluted under acidic conditions) are shown. It is a pie chart which shows the result of having classified the refined O-linked N-acetylglucosamine glycoprotein according to function using DAVID bioinformatics database. It is a photograph which shows the result of having analyzed the refined O-linked N-acetylglucosamine glycoprotein by Western blotting. It is a photograph of the gel which performed CBB dyeing | staining which shows the result of recombinant OGT protein (rec.OGT) refinement | purification. In FIG. 5, the arrow indicates the OGT protein. It is a photograph which shows the result of the in vitro OGT analysis using recombinant histone protein. The upper row in FIG. 6 shows autoradiography (Auto) in which each recombinant histone protein radiolabeled (N-acetylglucosamined) with UDP- [ ³ H-] GlcNAc and recombinant OGT was detected, and the lower row shows each The photograph (CBB) of the gel with which CBB staining was performed which detected the recombinant histone protein is shown. It is a photograph which shows the result of the in vitro OGT analysis using recombinant MLL5 protein. The left side in FIG. 7 shows autoradiography (Auto) detecting MLL5 radiolabeled (N-acetylglucosamined) with UDP- [ ³ H-] GlcNAc and recombinant OGT, and the right side detected MLL5. The photograph (CBB) of the gel to which CBB dyeing was given is shown. It is a photograph which shows the result of the in vitro OGT analysis using the histone octamer reconstituted in vitro. The upper row in FIG. 6 shows autoradiography (Auto) in which recombinant histone H2B radiolabeled (N-acetylglucosamined) with UDP- [ ³ H-] GlcNAc and recombinant OGT (rec. OGT) was detected. The lower row shows a photograph (CBB) of a gel subjected to CBB staining in which each recombinant histone protein was detected. It is a photograph which shows the result of the in vitro OGT analysis of N-acetylglucosamination of the histone octamer derived from Hela cells (Human) and Drosophila (Fly). The upper row in FIG. 9 shows autoradiography (Auto) in which recombinant histone H2B detected by radiolabeling (N-acetylglucosamine) with UDP- [ ³ H-] GlcNAc and recombinant OGT is detected, and the lower row shows each recombination. The photograph (CBB) of the gel which performed the CBB dyeing | staining which detected the histone protein is shown. In vitro N-acetylglucosamined histone octamer (0.5 μg) is digested with trypsin, concentrated to some extent with WGA lectin-conjugated magnetic beads, supernatant (first and third columns) and eluate (second And the fourth column) was subjected to LC-ETD-MS / MS and is a schematic view of the identification process of O-linked N-acetylglucosamine in a histone octamer. It is a spectrum figure which shows the result of the ETD-MS / MS scan (2349.43 m / z) of N-acetylglucosamine peptide. The amino acid numbers shown in the figure are the amino acid numbers counted from the first methionine. Results of ETD-MS / MS scan (m / z 731.6080 (3+)) of N-acetylglucosamined peptide, that is, peptide consisting of 78 to 94 amino acids of H2B to which mono GlcNAc is bound via the 91st serine FIG. The amino acid numbers shown in the figure are the amino acid numbers counted from the first methionine. Results of ETD-MS / MS scan (m / z 588.620 (4+)) of N-acetylglucosamined peptide, that is, peptide consisting of 95 to 114 amino acids of H2B to which mono GlcNAc is bound via the 112th serine FIG. The amino acid numbers shown in the figure are the amino acid numbers counted from the first methionine. Results of ETD-MS / MS scan (m / z 491.9090 (3+)) of N-acetylglucosamined peptide, that is, peptide consisting of 115 to 126 amino acids of H2B in which mono GlcNAc is bound via the 123rd serine FIG. The amino acid numbers shown in the figure are the amino acid numbers counted from the first methionine. It is a spectrum figure which shows the result of the quantitative TOF-MS analysis of in vitro N-acetylglucosamined H2B. The scan result of the O-linked N-acetylglucosamine site using the H2B peptide library described in Table 1 is shown. In addition, the upper part in FIG. 16 shows the position in the H2B peptide, and in the lower part, the vertical axis shows the amount normalized with respect to the control peptide of a similar Ala mutant (mutant). It is a spectrum figure which shows the result of the quantitative TOF-MS analysis of in vitro N-acetylglucosamined H2B partial peptide (101-115 amino acids). It is a spectrum figure which shows the result of the quantitative TOF-MS analysis of in vitro N-acetylglucosamined H2B partial peptide (111-125 amino acids). It is a figure which shows the recombinant H2B variant used for the in-vivo OGT analysis. The result of the in-vivo OGT analysis using the Xenopus H2B recombination deletion (H2B (delta) N) from which the N-terminal tail (N-terminal tail) is deleted is shown. The upper part in FIG. 20 shows autoradiography (Auto) in which each histone H2B detected by radiolabeling (N-acetylglucosamine) with UDP- [ ³ H-] GlcNAc and recombinant OGT is detected, and the lower part shows each histone. The photograph (CBB) of the gel which performed CBB dyeing | staining which detected H2B is shown. The result of the in vivo OGT analysis using the human-derived H2B recombinant mutant which substituted serine / threonine with the alanine is shown. 21 shows autoradiography (Auto) in which each histone H2B detected by radiolabeling (N-acetylglucosamination) with UDP- [ ³ H-] GlcNAc and recombinant OGT is detected. The photograph (CBB) of the gel which performed CBB dyeing | staining which detected H2B is shown. The result of the in vivo OGT analysis using the human-derived H2B recombinant mutant which substituted serine / threonine with alanine is shown. The upper row in FIG. 22 shows autoradiography (Auto) in which each histone H2B detected by radiolabeling (N-acetylglucosamine) with UDP- [ ³ H-] GlcNAc and recombinant OGT is detected, and the lower row shows each histone. The photograph (CBB) of the gel which performed CBB dyeing | staining which detected H2B is shown. It is a figure which shows the result of having compared the amino acid sequence of (alpha) helix ((alpha) C) of C terminal of H2B. In FIG. 23, “S” (the 112th and 123rd serine residues) labeled in black indicates an N-acetylglucosamination site, and the labeled “K” (the 120th lysine residue) indicates a ubiquitination site. Show. FIG. 2 is a structural diagram of a nucleosome showing the positions of H2B O-linked N-acetylglucosamine sites and ubiquitination sites in the nucleosome. In addition, the white ribbon in FIG. 24 shows αC helix. It is a figure which shows the partial peptide (peptide which consists of an amino acid sequence of sequence number 3) of the histone H2B by which N-acetylglucosamine of 112th serine was used as an antigen. It is a graph which shows the result of evaluation of (alpha) -H2B-S112-GlcNAc antibody (anti-histone H2B Ser112 GlcNAc antibody) by ELISA. As a negative control, a partial peptide of histone H2B (CKHAVSEGTK) that was not N-acetylglucosamined was used. It is a photograph which shows the result by the western blotting analysis using histone H2B and histone H2A (each 0.5 microgram) which carried out N-acetylglucosamine in vitro. It is a photograph which shows the result by the Western blotting analysis which used (alpha) -H2B-S112-GlcNAc antibody about the chromatin extract derived from a HeLa cell. The arrow indicates the histone H2B protein and the asterisk indicates a non-specific band. It is a photograph which shows the result of having analyzed the time-dependent change of O-linked N-acetylglucosamination and ubiquitination in histone H2B after glucose stimulation by Western blotting. It is a graph which shows the result of having analyzed the time-dependent change of O-linked N-acetylglucosamination and ubiquitination in histone H2B after glucose stimulation. In addition, the intensity | strength of the band of a western blotting was quantified by the ImageJ program. 1 is a schematic diagram showing the sequence of shRNA against OGT (shOGT) and the structure of the recombinant retrovirus, as well as infection and selection of the recombinant retrovirus. It is a photograph which shows the result of having analyzed the efficiency of OGT knockdown by shRNA by Western blotting. As a negative control, shRNA (shLuc) for luciferase was used. It is a photograph which shows the result of having analyzed the influence of OGT knockdown with respect to the glucose dependence up-regulation of N-acetylglucosamine of H2B by Western blotting. It is a photograph which shows the result of having analyzed the influence of OGT knockdown with respect to the glucose dependence up-regulation of N-acetylglucosamination and ubiquitination of H2B in HeLa cell by Western blotting. Glucose-free (Cont.), 1 mM pyruvate (Pyr), 10 mM GlcNAc, or 4.5 g / L-glucose (Glc) -containing DMEM, or 4.5 g / L-glucose (Glc) -containing DMEM and / or HBP inhibition Western blotting of O2-linked N-acetylglucosamine amount of H2B in HeLa cells cultured with an agent (6-diazo-5-oxo-L-norleucine (100M, DON) or azaserine (100M, AZA)) It is a photograph which shows the result analyzed by this. Western H2B AA mutation (histone H2B in which amino acids (S112 and T122) essential for N-acetylglucosamination are substituted with alanine) for glucose-dependent up-regulation of H2B N-acetylglucosamination and ubiquitination in HeLa cells It is a photograph which shows the result analyzed by blotting. It is a photograph which shows the result of having analyzed the interaction between histone H2B (WT) or H2B AA mutation and BRE1A by co-immunoprecipitation and Western blotting. It is a photograph which shows the result of having analyzed the glucose dependent interaction of histone H2B (WT) and BRE1A by the co-immunoprecipitation method and the western blotting. In the figure, “ cont. ” Indicates the results under glucose depletion conditions, and “ Glc ” indicates the results under glucose addition conditions. The photograph of the gel with which CBB dyeing which shows the result of the refinement | purification of recombinant ubiquitin ligase (rec.E1, rec.RAD6A, rec.BRE1A / 1B complex) was performed is shown. Each arrow points to the desired recombinant ubiquitin ligase. The photograph (middle stage and lower stage) which shows the result of having analyzed the in vitro ubiquitination assay using H2B and recombinant E1-RAD6A-BRE1A / 1B ubiquitin ligase by Western blotting, and the graph (upper stage) which digitized it are shown. Photographs (middle and lower panels) showing the results of analyzing in vitro ubiquitination assay using N-acetylglucosamined H2B by Western blotting using α-H2B-K120-ub and α-H2B antibodies, and quantifying it The graph (upper row) is shown. A strain having a sxc / ogt loss of function mutant (sxc ¹ ) or a wild-type fly (+ / +) is crossed with a fly of the In (1) W ^m4h strain and PEV (group position effect) ) Is a photograph of a fly compound eye showing the result of analyzing the histone modification by the color region of the offspring eye obtained by mating. It is a graph which shows the result of having measured the light absorbency (OD) in wavelength 480nm in the compound eye of each fly shown in FIG. In addition, the average value obtained by three independent measurements is shown with a standard error. It is a microscope picture which shows the result of having immunostained the wild-type multifilament chromosome of a fly by DAPI, (alpha) -H2B-S112-GlcNAc antibody, or (alpha) -H3K4me2 antibody (the 4th dimethylated lysine antibody of anti-histone H3). In the figure, the green light-emitting part indicates the part stained with the α-H2B-S112-GlcNAc antibody, and the red light-emitting part indicates the part stained with the α-H3K4me2 antibody. A portion emitting blue light indicates a portion stained with DAPI. “Merge” is a superposition of these stained photographs. Note that the fourth dimethylated lysine of histone H3 is a marker for the transcriptionally active chromatin region, and DAPI stains DNA. Fig. 45 is a photomicrograph showing the result of magnifying and observing a part of the wild-type multifilament chromosome of the fly shown in Fig. 44. “Merge [DAPI (−)]” is a superposition of a photograph of immunostaining with α-H2B-S112-GlcNAc antibody and a photograph of immunostaining with α-H3K4me2 antibody. It is a microscope picture which shows the result of having immunostained the wild type polytene chromosome of a fly by DAPI, (alpha) -H2B-S112-GlcNAc antibody, or (alpha) -H3K9me2 antibody (the 9th dimethylated lysine antibody of anti-histone H3). In the figure, the green light emitting part shows the part stained with the α-H2B-S112-GlcNAc antibody, and the red light emitting part shows the part stained with the α-H3K9me2 antibody. A portion emitting blue light indicates a portion stained with DAPI. “Merge” is a superposition of these stained photographs. Note that the 9th dimethylated lysine of histone H3 is a marker of the transcription repressing chromatin region, and DAPI stains DNA. It is a microscope picture which shows the result of having expanded and observed a part of wild type multifilament chromosome of the fly shown in FIG. “Merge [DAPI (−)]” is a superposition of a photograph of immunostaining with α-H2B-S112-GlcNAc antibody and a photograph of immunostaining with α-H3K9me2 antibody. It is a microscope picture which shows the analysis result of the intracellular distribution of N-acetylglucosaminated H2B by the immuno-staining using DAPI or (alpha) -H2B-S112-GlcNAc antibody. “Merge” is a superposition of a photograph stained with DAPI and a photograph photographed with the α-H2B-S112-GlcNAc antibody. It is a pie chart which shows the result of the genome wide distribution analysis of N-acetylglucosamined H2B site. It is a graph which shows the correlation with the transcriptional activity in an N-acetylglucosaminated H2B site | part analyzed by the microarray using the total RNA obtained from HeLa cell, and each gene. It is a graph which shows the average profile in the vicinity of TSS (transcription start position) and TTS (transcription end position), or the whole gene of ChIP region. It is a figure which shows the transcription factor (HNF4) binding site in the N-acetylglucosamination site of H2B. The notation is based on the TRANSFAC library. It is a figure which shows the transcription factor (COUPTF) binding site in the N-acetylglucosamination site of H2B. The notation is based on the TRANSFAC library. It is a graph which shows the analysis result by ChIP-seq and qPCR using the promoter region of PGK1 which is a glucose responsive gene. In the upper part of the figure, the promoter region of PGK1 and the position of the primer set used for the analysis are shown. In addition, ChIP (chromatin immunoprecipitation) is performed using α-H2B-S112-GlcNAc antibody, α-H3K4me3 antibody (anti-histone H34th trimethylated lysine antibody), α-H2BK120Ub antibody, and α-AcH3 antibody (anti-acetylated). Histone H3 antibody), and α-H2B and α-H3 antibodies. The vertical axis | shaft of each graph in the figure shows the ratio of the histone protein which is glycosylated, ubiquitinated, or acetylated in histone H2B or histone H3 in each region. In the figure, the black bar graph shows the ratio of each histone modification after glucose stimulation (Glc), and the white bar graph shows the ratio of each histone modification before glucose stimulation (Cont.). It is the schematic about the function of N-acetylglucosaminated H2B in an O-GlcNAc signal transduction process. Glucose is metabolized to UDP-GlcNAc by HBP (hexamine biosynthetic pathway) branched from the glycolytic pathway. Since OGT activity depends on the intracellular concentration of UDP-GlcNAc, the degree of O-GlcNAc modification of H2B reflects extracellular glucose. N-acetylglucosamined H2B is a target of the BRE1 complex that ubiquitinates lysine at the 120th amino acid residue adjacent to it. The simultaneous activation in the C-terminal α-helix of H2B between the O-GlcNAc modification of the 112th serine and the ubiquitination of the 120th lysine is a sequential modification such as H3K4 methylation required for transcription initiation. Trigger the reaction. The concept of these signal transduction processes is illustrated.

<Method for detecting glycosylation of histone protein>
The method for detecting glycosylation of the histone protein of the present invention comprises the following steps (a) to (b):
(A) A step of preparing a sample containing a histone protein (b) A method of detecting a glycosylated histone protein in the sample.

  In the present invention, the “histone protein” refers to a core histone protein (H2A, H2B, H3, H4) and a linker histone protein (H1) constituting a nucleosome that is a basic unit of chromatin. The histone protein in the present invention includes these variants, such as H2AX and H2AZ.

  “Glycosylation” in the present invention refers to glycosylation on histones. The type of sugar constituting the sugar chain is not particularly limited, and examples thereof include glucose, galactose, mannose, fucose, N-acetylglucosamine, N-acetylgalactosamine, N-acetylneuraminic acid, and xylose. The modification is not particularly limited, and for example, O-linked glycosylation for adding a sugar chain to a serine or threonine residue in histone, and N-linked glycosyl to add a sugar chain to an asparagine residue in histone Can be mentioned.

  In addition, the “sample” used in the present invention is not particularly limited, and examples thereof include cells, tissues, extracts containing histone proteins therefrom, or purified or crudely purified histone proteins. Preparation of an extract containing a histone protein or a purified or crude purified histone protein can be performed by appropriately adopting known methods. For example, the method as described in the following Example <Biochemical purification and identification of N-acetylglucosamined glycoprotein> can be given.

  As a method of “detecting a glycosylated histone protein” in the present invention, a glycosylated histone protein in a sample prepared in the above-mentioned “preparing a sample containing a histone protein” can be detected. Any known method can be adopted as appropriate. For example, a method using mass spectrometry as described in Examples <In vitro N-acetylglucosamination analysis (mass spectrometry)>, or <In vitro N-acetylglucosamination analysis (western blotting analysis)> Western blotting using antibodies that bind to sugars or glycosylated sites that constitute simple sugar chains, immunoprecipitation using these antibodies, and examples described below <In vitro N-acetylglucosamine analysis (Autoradiographic analysis)> A method for detecting a histone protein glycosylated by autoradiography as shown in FIG. Among these, from the viewpoint of detection specificity and convenience, a method using an antibody that binds to a sugar constituting a sugar chain or a glycosylated site is more preferable. Furthermore, from the viewpoint that it can be detected with higher specificity than an antibody that binds to a sugar constituting the sugar chain and is less susceptible to steric hindrance at the glycosylated site, the glycosyl of histone protein. Preferably, the method uses an antibody that binds to the activated site.

  The glycosylation of the histone protein to be detected is not particularly limited, but N-acetylglucosamine is preferable. Furthermore, the glycosylated histone protein to be detected is not particularly limited, but is preferably a glycosylated histone H2A and / or histone H2B.

  When the detected glycosylated histone protein is histone H2B, at least one serine residue selected from the group consisting of the 91st serine residue, the 112th serine residue, and the 123rd serine residue is used. The group is preferably glycosylated histone H2B, and the 112th serine residue is more preferably N-acetylglucosamined histone H2B.

<Method for evaluating serum glucose level>
As shown in the Examples below, the amount of glycosylated histone protein depends on the extracellular glucose concentration via the hexosamine biosynthetic pathway (HBP), so that histone protein glycosylation is associated with serum glucose levels. It becomes an index for detecting. Accordingly, the present invention provides a method for evaluating serum glucose level, comprising detecting a glycosylated histone protein comprising the following steps (a) to (b): (a) (B) A step of detecting a glycosylated histone protein in the sample.

  In the present invention, “serum glucose level” means the level or concentration of glucose in blood, and is a concept that includes blood glucose level and plasma glucose level.

  The “glycosylated histone protein” to be detected is not particularly limited as long as it is a histone protein whose glycosylation is enhanced depending on the extracellular glucose concentration via HBP. Insulin dependence) From the viewpoint that the relationship between diabetes and abnormal expression (enhancement) has been clarified ("Folli et al., PLoS One, March 29, 2010, Vol. 3, No. 3, e9923" and "Suhail et al , Indian L Med Res, February 1990, Vol. 92, pp. 21-23), preferably a glycosylated histone protein derived from the phosphoglycerate kinase 1 (PGK1) gene.

  The “sample”, “method for preparing a sample containing histone protein”, and “method for detecting glycosylated histone protein” are the same as the above <Method for detecting glycosylation of histone protein>. is there.

<Method for examining metabolic syndrome>
As described above, since the amount of glycosylated histone protein reflects the amount of serum glucose, it is possible to examine metabolic syndrome using the glycosylation of histone protein as an index. Accordingly, the present invention also provides a method for examining metabolic syndrome characterized by detecting a glycosylated histone protein comprising the following steps (a) to (b): (B) A step of detecting a glycosylated histone protein in the sample.

  The “metabolic syndrome” in the present invention may be any disease or symptom caused by an increase in the serum glucose level. For example, hyperglycemia, hyperlipidemia, hypertension, diabetes (type 1 and type 2 diabetes), arteriosclerosis Cerebral infarction, stroke, myocardial infarction, heart failure.

  “Sample”, “Method of preparing sample containing histone protein”, “Method of detecting glycosylated histone protein”, and “glycosylated histone protein” to be detected are <glycosyl of histone protein> This is the same as <Method for detecting oxidization> and <Method for evaluating serum glucose level>.

  If the detection by this method results in a greater amount of glycosylated histone protein than a control (eg, that amount in a healthy subject), the subject is assessed as having or suspected of having metabolic syndrome can do.

<Method for screening drug candidate compounds against metabolic syndrome>
As described above, since the amount of glycosylated histone protein reflects the amount of serum glucose, it is possible to develop a drug for metabolic syndrome using the glycosylation of histone protein as an index. Accordingly, the present invention also provides a method for screening a drug candidate compound against metabolic syndrome, comprising the following steps (a) to (d): (a) a step of preparing a sample containing histone protein (b) ) Contacting the test compound with the sample (c) detecting glycosylated histone protein in the sample contacted with the test compound (d) comparing with not contacting the test compound in (c) Selecting a compound that reduces the amount of glycosylated histone protein detected.

  In the screening method of the present invention, the “test compound” to be brought into contact with the sample containing histone protein is not particularly limited. For example, the expression product of a gene library, a synthetic low molecular compound library, a peptide library, an antibody , Bacterial release materials, extracts of cells (microorganisms, plant cells, animal cells) and culture supernatants, purified or partially purified polypeptides, extracts from marine organisms, plants or animals, soil, random phage peptide display libraries Can be mentioned.

  "Sample", "Method for preparing sample containing histone protein", "Method for detecting glycosylated histone protein", "Glycosylated histone protein" to be detected, and "Metabolic syndrome" It is the same as <Method for detecting glycosylation of histone protein>, <Method for evaluating serum glucose level>, and <Method for examining metabolic syndrome>.

  As shown in the Examples below, the amount of glycosylated histone protein reflects the extracellular glucose concentration via the hexosamine biosynthetic pathway (HBP). It means that the activation of signal transduction accompanying an increase in the external glucose concentration is suppressed. In addition, as shown in the examples described later, since the glycosylation of histone protein activates transcription of PGK1 gene and the like that are contributed to diabetes, if the amount of glycosylated histone protein decreases. Normalization of gene or protein expression that has become abnormal with increasing extracellular glucose concentration can result.

  Therefore, when the amount of glycosylated histone protein detected in (c) is decreased as a result of detection compared to the case where the test compound is not contacted, the test compound is a drug candidate compound for metabolic syndrome. Can be selected.

<Antibodies that bind to glycosylated sites of histone proteins>
The present invention provides antibodies that bind to glycosylated sites of histone proteins. “Glycosylation” to be detected by the antibody of the present invention is not particularly limited, but N-acetylglucosamine is preferable. Furthermore, the “histone protein” is not particularly limited, but is preferably histone H2A and / or histone H2B. When the glycosylated histone protein is histone H2B, at least one selected from the group consisting of 91st serine residue, 112th serine residue, and 123rd serine residue of histone H2B. The serine residue is preferably a glycosylated site, more preferably the 112th serine residue of histone H2B is a site that is N-acetylglucosamined.

  The “antibody” of the present invention may be a polyclonal antibody, a monoclonal antibody, or a functional fragment of an antibody. “Antibodies” also includes all classes and subclasses of immunoglobulins. “Polyclonal antibodies” are antibody preparations comprising different antibodies directed against different epitopes. The “monoclonal antibody” means an antibody (including an antibody fragment) obtained from a substantially homogeneous antibody population. In contrast to polyclonal antibodies, monoclonal antibodies are those that recognize a single determinant on an antigen. The antibodies of the invention are antibodies that have been separated and / or recovered (ie, isolated) from components of the natural environment. In the present invention, the “functional fragment” of an antibody means a part (partial fragment) of an antibody that specifically recognizes a target protein. Specifically, Fab, Fab ′, F (ab ′) 2, variable region fragment (Fv), disulfide bond Fv, single chain Fv (scFv), sc (Fv) 2, diabody, multispecific antibody, And polymers thereof.

  If the antibody of the present invention is a polyclonal antibody, an immunized animal is immunized with an antigen (such as a target glycosylated histone protein, a partially glycosylated peptide thereof, or a cell expressing these), and the antiserum is subjected to conventional means ( For example, it can be purified and obtained by salting out, centrifugation, dialysis, column chromatography, etc.). Monoclonal antibodies can be prepared by a hybridoma method or a recombinant DNA method.

  The hybridoma method typically includes the method of Kohler and Milstein (Kohler & Milstein, Nature, 256: 495 (1975)). The antibody-producing cells used in the cell fusion step in this method are animals (eg, mice, rats, hamsters) immunized with an antigen (such as a target glycosylated histone protein, a partially glycosylated peptide thereof, or a cell expressing them). Rabbits, monkeys, goats) spleen cells, lymph node cells, peripheral blood leukocytes and the like. It is also possible to use antibody-producing cells obtained by allowing an antigen to act on the above-mentioned cells or lymphocytes previously isolated from an unimmunized animal in a medium. As the myeloma cells, various known cell lines can be used. The antibody-producing cells and myeloma cells may be of different animal species as long as they can be fused, but are preferably of the same animal species. The hybridoma is produced, for example, by cell fusion between a spleen cell obtained from a mouse immunized with an antigen and a mouse myeloma cell, and a hybridoma that produces a monoclonal antibody specific for the target protein is obtained by subsequent screening. Obtainable. A monoclonal antibody against the target protein can be obtained by culturing the hybridoma or from the ascites of the mammal to which the hybridoma has been administered.

  In the recombinant DNA method, a DNA encoding the antibody or peptide of the present invention is cloned from a hybridoma, a B cell or the like and incorporated into an appropriate vector, which is then introduced into a host cell (for example, a mammalian cell line, E. coli, yeast cell, insect). Cells, plant cells, etc.), and the antibody of the present invention is produced as a recombinant antibody (for example, PJ Delves, Antibody Production: Essential Technologies, 1997 WILEY, P. Shepherd and C. Dean Monoclonal). Antibodies, 2000 OXFORD UNIVERSITY PRESS, Vandame AM et al., Eur. J. Biochem. 192: 767-775 (1990)). In the expression of the DNA encoding the antibody of the present invention, DNA encoding the heavy chain or light chain may be separately incorporated into an expression vector to transform the host cell. Host cells may be transformed into a single expression vector (see WO94 / 11523). The antibody of the present invention can be obtained in a substantially pure and uniform form by culturing the above host cell, separating and purifying it from the host cell or culture medium. For the separation and purification of the antibody, the methods used in the usual purification of polypeptides can be used. If transgenic animals (such as cows, goats, sheep or pigs) in which an antibody gene is incorporated are produced using transgenic animal production technology, a large amount of monoclonal antibody derived from the antibody gene is produced from the milk of the transgenic animal. It is also possible to obtain.

  Amino acid sequence variants of the antibodies of the invention can be made by introducing mutations into DNA encoding the antibody chain or by peptide synthesis. The site where the amino acid sequence of the antibody is modified may be the constant region of the heavy chain or light chain of the antibody as long as it has an activity equivalent to that of the antibody before modification, and the variable region (framework region and CDR). Modification of amino acids other than CDR is considered to have a relatively small effect on the binding affinity with the antigen, but at present, the amino acid of the CDR is modified to screen for an antibody having an increased affinity for the antigen. Methods are known (PNAS, 102: 8466-8471 (2005), Protein Engineering, Design & Selection, 21: 485-493 (2008), International Publication No. 2002/051870, J. Biol. Chem., 280: 24880- 24887 (2005), Protein Engineering, Design & Selection, 21: 345-351 (2008)).

  The number of amino acids to be modified is preferably within 10 amino acids, more preferably within 5 amino acids, and most preferably within 3 amino acids (eg, within 2 amino acids, 1 amino acid). The amino acid modification is preferably a conservative substitution. In the present invention, “conservative substitution” means substitution with another amino acid residue having a chemically similar side chain. Groups of amino acid residues having chemically similar amino acid side chains are well known in the technical field to which the present invention belongs. For example, acidic amino acids (aspartic acid and glutamic acid), basic amino acids (lysine, arginine, histidine), neutral amino acids, amino acids with hydrocarbon chains (glycine, alanine, valine, leucine, isoleucine, proline), hydroxy groups Amino acids with amino acids (serine / threonine), amino acids with sulfur (cysteine / methionine), amino acids with amide groups (asparagine / glutamine), amino acids with imino groups (proline), amino acids with aromatic groups (phenylalanine / tyrosine / (Tryptophan). The amino acid sequence variant preferably has an antigen-binding activity equivalent to that of a control antibody (for example, the antibody α-H2B-S112-GlcNAc antibody described in this Example). The binding activity to the antigen can be evaluated, for example, by ELISA, Western blotting, immunoprecipitation, or immunostaining.

<Agent for detecting glycosylated histone protein, agent for evaluating serum glucose level, and agent for examining metabolic syndrome>
As described above, an antibody that binds to a glycosylated site of such histone protein is obtained by a method for detecting glycosylation of the histone protein of the present invention, a method for evaluating serum glucose level, and a pharmaceutical against metabolic syndrome. It can be suitably used for “detection of glycosylated histone proteins” in the method for screening candidate compounds. Therefore, the present invention provides an agent for detecting glycosylated histone protein, an agent for evaluating serum glucose level, and an agent for examining metabolic syndrome, comprising the antibody of the present invention as an active ingredient. .

  As an antibody used as an active ingredient of the drug of the present invention, an antibody to which a labeling substance is bound can be used. By detecting the label, the amount of antibody bound to the target protein can be directly measured. The labeling substance is not particularly limited as long as it can bind to an antibody and can be detected by a chemical or optical method. For example, peroxidase, β-D-galactosidase, microperoxidase, horseradish peroxidase (HRP), fluorescein isothiocyanate (FITC), rhodamine isothiocyanate (RITC), alkaline phosphatase, biotin, and radioactive substances.

  In addition to directly measuring the amount of antibody bound to the target protein using an antibody bound to the labeling substance, a method using a secondary antibody bound to the labeling substance or a method of binding the secondary antibody to the labeling substance. An indirect detection method such as a method using a polymer can also be used. Here, the “secondary antibody” is an antibody exhibiting specific binding to the antibody of the present invention. For example, when the antibody of the present invention is prepared as a rabbit antibody, an anti-rabbit IgG antibody can be used as the secondary antibody. Labeled secondary antibodies that can be used are commercially available for antibodies derived from various biological species such as rabbits, goats, and mice, and appropriate secondary antibodies are available depending on the biological species from which the antibody of the present invention is derived. Can be selected and used in the present invention. Instead of the secondary antibody, protein G or protein A to which a labeling substance is bound can be used.

  Furthermore, the agent of the present invention may contain other components acceptable as a composition in addition to the antibody component. Examples of such other components include carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, stabilizers, preservatives, preservatives, physiological saline, and the like. As the excipient, lactose, starch, sorbitol, D-mannitol, sucrose and the like can be used. As the disintegrant, starch, carboxymethylcellulose, calcium carbonate and the like can be used. Phosphate, citrate, acetate, etc. can be used as the buffer. As the emulsifier, gum arabic, sodium alginate, tragacanth and the like can be used. As the suspending agent, glyceryl monostearate, aluminum monostearate, methyl cellulose, carboxymethyl cellulose, hydroxymethyl cellulose, sodium lauryl sulfate and the like can be used. As the stabilizer, propylene glycol, diethylin sulfite, ascorbic acid or the like can be used. As preservatives, phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, methylparaben, and the like can be used. As a preservative, sodium azide, benzalkonium chloride, paraoxybenzoic acid, chlorobutanol and the like can be used.

  In addition to the agent of the present invention, a substrate necessary for detection of a label, a positive control or a negative control, or a buffer used for dilution or washing of a sample can be combined to detect glycosylated histone protein. It can also be a kit or the like. Further, when an unlabeled antibody is used as an antibody preparation, those labeled with a substance that binds to the antibody (for example, secondary antibody, protein G, protein A, etc.) can be combined. Furthermore, a kit for detecting a glycosylated histone protein or the like can include instructions for using the kit.

  EXAMPLES Hereinafter, although this invention is demonstrated more concretely based on an Example, this invention is not limited to a following example. Moreover, each Example was performed in accordance with the method described below.

<Cell preparation method>
HeLa cells were maintained in DMEM containing 10% FBS and subjected to the following gene transfer and the like.

  In order to prepare a cell that stably expresses H2B or a mutant thereof to which a FLAG tag sequence is bound, first, cDNA of H2B having a FLAG tag sequence bound to the 5 ′ end, and a mutant thereof are designated as pcDNA3 (Invitrogen). Subcloning into the product). A series of H2B partial mutants were subcloned into pET3 vector (manufactured by Novagen) and prepared according to a basic mutant preparation method. Then, pcDNA vectors encoding FLAG tag binding H2B or mutant H2B were introduced into HeLa cells, and cells each containing these vectors were selected by culturing under conditions of 0.5 mg / mL G418.

In order to prepare a cell in which OGT is knocked down, first, an shRNA sequence (5′-GCACATACACATCTGGCTTCC-3 ′: SEQ ID NO: 1) targeting hOGT and an shRNA sequence (5′-TGCGTTGCTAGTACCAACAC) targeting Renilla Luc. -3 '
: SEQ ID NO: 2, non-target control) was inserted into each pSIREN-RetroQ-ZsGreen vector (Clontech). Furthermore, for the production of retrovirus, the constructed shRNA vector was introduced into PLAT-A cells using Lipofectamine 2000 solution (Invitrogen). 24 hours after the introduction, the culture medium was replaced with DMEM (Dulbecco's modified Eagle) medium containing 10% FBS (fetal bovine serum), and then the cells were cultured for 24 hours. The supernatant of the medium containing the virus was used for infection. In addition, infection, selection of infected cells and the like were performed according to the method described in Non-Patent Document 22. That is, in order to create a OGT-KD (knockdown) cells by retroviral infection, seeded with 60 mm 10 ⁶ cells in culture dishes, retroviral cocktail 3 ml (1 ml: prepared retroviral solution, 2 ml: DMEM medium containing 10% FBS and 8 g / mL polybrene) and further cultured for 48 hours. Then, using a FACS Vantage (BD) classification device, eGFP positive cells infected with retrovirus were separated. The expression level and knockdown efficiency were confirmed by immunoblotting and immunofluorescence staining.

  In addition, in order to control the amount of N-acetylglucosamine in the nucleus, the cells are first cultured in a 4.5 g / L glucose-containing medium (15 minutes to 24 hours) and then in a DMEM medium not containing glucose for 24 hours. The cells were stimulated by depleting glucose and used for the next experiment as appropriate.

<Method for producing monoclonal antibody>
A partial peptide of histone H2B [CKHASV S (GlcNAc) EGTK: SEQ ID NO: 3] in which the 112th serine is N-acetylglucosamined was obtained from MBL Institute, Inc. And used as an antigen to immunize mice. Then, using this antigen, monoclonal antibody-producing cells (hybridomas) are prepared by Operan Biotechnologies, and the obtained hybridomas are simply screened together with O-linked N-acetylglucosamine peptides using ELISA, and in vitro O -Sorted by immunoblot analysis with conjugated N-acetylglucosamined H2B.

<Immunoprecipitation and Western blotting>
Immunoprecipitation and Western blotting (immunoblot) were performed according to the method reported by the inventors. That is, the chromatin component was extracted from the nuclear precipitate fraction by 4 cycles of sonication. The immunoprecipitate by chromatin extract or α-FLAG M2 agarose (manufactured by Sigma) was analyzed by Western blotting using the following antibodies.
α-H2A, α-H2B, α-H3, α-H4 (manufactured by Abcam)
α-H2B-K120-ub (manufactured by Upstate)
α-O-GlcNAc (RL2 or CTD110.6, manufactured by Abcam)
α-OGT (manufactured by Sigma)
α-FLAG (manufactured by Sigma)
α-H2B-S112-GlcNAc (antibody prepared by ordering from Operon shown in FIGS. 25 to 27)
α-MLL5 (N) (manufactured by Operon, see Non-Patent Document 22)
α-RNF20 / BRE1A (Bethyl).

<Production of recombinant protein>
The recombinant protein was prepared according to the methods described in Non-Patent Documents 11 and 12. That is, recombinant FLAG-OGT, FLAG-E1, and FLAG-BRE1A / B complexes are expressed using baculovirus and isolated by purification mainly using immunoprecipitation using α-FLAG M2 agarose (manufactured by Sigma). did. Recombinant 6 × His-RAD6A was expressed in bacteria and isolated to some extent using HIS-Select Nickel Affinity Gel (manufactured by Sigma). The eluate was diluted so that the eluate: BCO [20 mM HEPES, 0.2 mM EDTA, 10% (v / v) glycerol, pH 7.9] was 1:20, and Rsource Q column (manufactured by GE Healthcare) was used. And fractionated by a linear concentration gradient (0-0.5 M KCl).

  Recombinant Xenopus histone H2B and its variants were prepared according to the method reported by the present inventors. That is, these proteins were expressed independently in BL21 (DE3) pLysS (manufactured by Promega), and these inclusion bodies were simply fractionated. Precipitated histones were solubilized in DMSO and 7M guanidinium-HCl, further purified through HiTrap Q HP (GE Healthcare) and bound to HiTrap SP HP (GE Healthcare). Bound histones were eluted with a linear concentration gradient (0.1-0.4M NaCl, 7M urea buffer).

<Biochemical purification and identification of N-acetylglucosamined glycoprotein>
To purify O-linked N-acetylglucosamine glycoprotein, 15 μg α-O-GlcNAc (RL2) antibody (Abcam) was added to 0.5 mL Dynabeads M-280 Sheep α-Mouse IgG (Invitrogen). ) According to the product instructions.

A chromatin extract (0.5 g protein) from HeLa cells was basically prepared according to the method described in Non-Patent Document 14. That is, a chromatin precipitate is obtained as a residue of a nuclear extract prepared using a buffer containing 1 mM streptozotocin (STZ), and is obtained as a micrococcal nuclease (MNase) buffer [20 mM Tris-HCl, 1 mM CaCl ₂ , 2 mM MgCl ₂ , 0 1 M KCl, 0.1% (v / v) Triton-X, 0.3 M sucrose, 1 mM DTT, 1 mM benzamide, 0.2 mM PMSF, 1 mM STZ, pH 7.9]. After adding 3 U / mL MNase, the sample was incubated for 30 minutes at room temperature with continuous homogenization, and then the reaction was stopped by adding 5 mM EGTA and 5 mM EDTA. After centrifugation at 2000 × g for 30 minutes at 4 ° C., a supernatant (chromatin extract) was obtained.

The obtained chromatin extract was passed through a WGA lectin agarose column (manufactured by Vector), and the effluent fraction was further mixed with beads immobilized with the α-O-GlcNAc antibody, followed by stirring at 4 ° C. for 8 hours. Buffer D [20 mM Tris-HCl, 0.2 mM EDTA, 5 mM MgCl ₂ , 0.1 M KCl, 0.05% (v / v) NP-40, 10% (v / v) glycerol, 1 mM DTT, 1 mM benzamide, After washing three times with 0.2 mM PMSF, 1 mM STZ, pH 7.9], the glycoprotein was eluted twice with buffer D supplemented with 0.4 mg / mL GlcNAc-O-Ser (MBL). (Elution # 1 and # 2) and finally eluted with 0.1 M glycine-HCl (pH 2.0) (elution # 3). The eluted protein was desalted by methanol-chloroform precipitation, digested with trypsin (manufactured by Promega), and an automatic LC-MS / MS apparatus (LC18ist, Zaprous nano-LC (AMR) piped with a C18 ESI reverse phase column). ), Finnegan LTQ ion-trap mass spectrometer (Thermo)). LC-MS / MS data were processed using Thermo BioWorks (Thermo) and SEQUEST (Thermo) to identify proteins. The identified protein was further analyzed using “gene functional classification tool” (DAVID bioinformatics resources 6.7 (http://david.abcc.ncifcrf.gov/)).

<In vitro N-acetylglucosamination analysis (Western blotting analysis)>
Recombinant OGT was incubated with free histones, in vitro associated histone octamers. The efficiency of H2B N-acetylglucosamine was analyzed by WB using an α-O-linked N-acetylglucosamine (RL2) antibody.

<In vitro N-acetylglucosamination analysis (autoradiographic analysis)>
Recombinant FLAG-OGT protein (0.5 μg) was combined with 0.5 μg of recombinant histone and 0.2 mM (0.2 μCi) of UDP- [ ³ H] GlcNAc (PerkinElmer) in 25 μL of reaction solution (50 mM Tris-HCl, 12.5 mM MgCl ₂ , 1 mM DTT, pH 7.5) and incubated at 37 ° C. for 24 hours. The reaction solution was separated using SDS-PAGE, blotted onto a PVDF membrane, sprayed with EN ³ HANCE (manufactured by NEN Lifescience), and then analyzed by autoradiography.

In addition, a core histone octamer (0.5 μg) derived from human Hela cells and fly S2 cells, a recombinant OGT protein (0.5 μg) and UDP- [ ³ H-] GlcNAc (0.2 μCi) at 37 ° C. The reaction was allowed for 10 hours. The obtained reaction product was developed by SDS-PAGE as described above, and electrotransfer was performed on the membrane. The reacted histone octamer was visualized by CDD staining and subjected to autoradiographic analysis.

<In vitro N-acetylglucosamination analysis (mass spectrometry)>
Recombinant histone (1 μg) or recombinant histone octamer (1 μg) reconstituted in vitro was prepared by recombinant FLAG-OGT using a 25 μL reaction solution (50 mM Tris-HCl, 2 mM UDP-GlcNAc, 12.5 mM MgCl ₂ , 1 mM DTT, N-acetylglucosamined at 37 ° C. for 24 hours at pH 7.5). The reaction solution was analyzed using a nano-LC ESI-TOF mass spectrometer (1100 nanoLC (Agilent), microCROF (Bruker)) piped with a ZORBAX 300SB-C18 column (Agilent). Further, the reaction solution was divided by trypsin (manufactured by Promega), and the glycopeptide was purified using MB-LAC WGA kit (manufactured by Bruker). The concentrated glycopeptide was analyzed using a nano-LC ESI-ETD ion trap mass spectrometer (Agilent HP1200 Nano (Agilent), amaZon ETD (Bruker)) piped with ZORBAX 300SB-C18 (Agilent). .

<In vitro ubiquitination analysis>
N-acetylglucosamined histone (1 μg) is 50 mM Tris (pH 7.9) using E1 (0.1 μg), RAD6 (0.2 μg), BRE1 complex (0.5 μg), ubiquitin (3 μg), Ubiquitination was carried out at 37 ° C. for 24 hours in 5 mM MgCl ₂ , 4 mM ATP. The obtained reaction product was developed by SDS-PAGE and subjected to Western blotting analysis.

<ChIP-Seq and ChIP-qPCR>
The ChIP and ChIP-Seq libraries were constructed according to the method described in “He et al., Nat Genet, 2010, Vol. 42, No. 4, pages 343-347”. In addition, the base sequence (35 base pairs) of the obtained library was determined using Genome Analyzer (manufactured by Illumina). Furthermore, the fragment of interest in the library was quantified by qPCR using a specific promoter set, Thermal Cycler TP800 (manufactured by TAKARA) and SYBR Premix Ex Taq II (manufactured by TAKARA). The specific promoter set is as follows.
(For peak 1082PGK1 gene)
5′-CGGACGGTGACAAACGGAAG-3 ′
: SEQ ID NO: 4
5'-CTATTGGCCACAGCCCATC-3 '
: SEQ ID NO: 5
(Exon 3 of PGK1 gene)
5'-ACAATGGAGCCAAGTCGGTAG-3 '
: SEQ ID NO: 6
5'-TTGCCCAGCAGAGATTTGAG-3 '
: SEQ ID NO: 7
(Exon 5 of PGK1 gene)
5′-CCGAGCCAGCCAAAATAGAAG-3 ′
: SEQ ID NO: 8
5'-TGAGCAGTGCCCAAAAGCATC-3 '
: SEQ ID NO: 9
(Exon 7 of PGK1 gene)
5′-AGTTGCAGACAAGATCCAGCTC-3 ′
: SEQ ID NO: 10
5'-TCACTACTGGCATTTGTTTCC-3 '
: SEQ ID NO: 11
(Exon 9 of PGK1 gene)
5′-GATTTGTGTGGAATGGTCCTTG-3 ′
: SEQ ID NO: 12
5′-AAGTGGCTTTCACCACCCTCATC-3 ′
: SEQ ID NO: 13
(PGK1 gene transcription end position (TTS)
5′-AGGAGAATGGCGGTGAACCTG-3 ′
: SEQ ID NO: 14
5'-CACACATGGGTTAGCTCTTTATGC-3 '
: SEQ ID NO: 15
The correspondence between each primer set and the PGK1 gene is shown in FIG.

Example 1
<Search for O-linked N-acetylglucosamined glycoprotein in the nucleus>
In order to search for N-acetylglucosamined glycoproteins in unknown nuclei that contribute to chromatin reorganization, immunoaffinity purification using specific antibodies capable of detecting O-linked N-acetylglucosamined sites was performed. went. That is, since O-linked N-acetylglucosamine of protein is caused by the presence of an excessive N-acetylglucosamine precursor, first, HeLa cells are cultured under high glucose conditions, and chromatin extract is obtained from such cells. And that the occurrence of excessive O-linked N-acetylglucosamine was confirmed by Western blotting using an anti-O-linked N-acetylglucosamine monoclonal antibody (RL2 antibody) (see FIG. 1). ). Then, the chromatin extract was purified stepwise using a WGA agarose column and magnetic beads coated with an immobilized anti-O-linked N-acetylglucosamine monoclonal (RL2) antibody (see FIG. 2). The purified product was digested with trypsin, and the trypsin-treated peptide was subjected to proteome analysis by liquid chromatography (LC) -mass spectrum (MS) / MS analysis. A total of 284 factors were identified, including the previously reported O-linked N-acetylglucosamine glycoproteins that function in RNA processing, translation, and nuclear transport (see FIG. 3).

  On the other hand, when the purified product is developed by SDS-PAGE and analyzed using silver staining, it is clear that the histone protein constituting the histone octamer can also be an O-linked N-acetylglucosamine glycoprotein. (See Fig. 2). Although endogenous histones could not be detected using the RL2 antibody (not shown in the figure), the presence of O-linked N-acetylglucosamine glycoprotein constituting histone octamers It became clear from the analysis by blotting (see Fig. 4).

(Example 2)
<O-linked N-acetylglucosamination of purified histone>
OGT is the only enzyme known to do O-linked N-acetylglucosamination of proteins in the nucleus (“Dong et al., J Biol Chem, 2004, 279, 3, 1577). Page ”), whether histone protein is a substrate of OGT. First, in vitro enzyme analysis was performed using recombinant human OGT (see FIG. 5) and histone protein (see the bottom of FIG. 6). The obtained result is shown in FIG. As is clear from the results shown in FIG. 6, histones H2A and H2B and histone H2A mutants (H2A.X and H2A.Z) have already been confirmed by the present inventors to be substrates of OGT5 ( Similar to FIG. 7, it was revealed that it was glycosylated in vitro by OGT.

  Next, since the substrate specificity of the enzyme that modifies histones differs between histone proteins present in the octamer and histone proteins present in a single subunit, the histone octamer is used. In vitro enzyme analysis was performed. The obtained results are shown in FIGS. As is clear from the results shown in FIGS. 8 and 9, in the histone octamer, the histone H2B was exposed on the surface of the histone octamer and was glycosylated by OGT (see FIG. 8). In addition, it was revealed that histone H2B is also N-acetylglucosamined in the associated Drosophila-derived histone octamer (see FIG. 9), and O-linked N-acetylglucosamine modification of histone H2B is a metazoan. Has also been demonstrated to occur.

(Example 3)
<Search for N-acetylglucosamination site by OGT in histone H2B>
Next, since the consensus sequence of the monosaccharide addition moiety mediated by OGT was not known (see Non-Patent Document 19), it was impossible to search related databases (“Kaleem et al., J Cell Biochem, 2008). 103, No. 3, p. 835), two different approaches (mass spectrometry and in vitro OGT analysis using H2B peptide library) determine the position of the O-linked N-acetylglucosamine site in the H2B protein. Decided to do.

  First, by mass spectrometry, the electron transfer dissociation (ETD) -MS / MS of the N-acetylglucosamined H2B histone octamer is 91 to a position shifted from the theoretical mass value by +203 Da (expected mass of the N-acetylglucosamine moiety). It was revealed that there are peaks of the 1st serine, the 112th serine and the 123rd serine (see FIGS. 10 to 14). In addition, in the quadrupole (Q) -TOF MS evaluation, it seems that the addition of monosaccharide has an influence when compared quantitatively with a di- or tri- moiety (N-acetylglucosaminated moiety) ( It was revealed that OGT can simultaneously transfer up to three N-acetylglucosamines to histone H2B.

  Next, in vitro OGT analysis using the H2B peptide library 101-115 in the C-terminal α helix (αC) of H2B by in vitro OGT (see Table 1) using a 15-mer peptide array covering the entire length of H2B or Two peaks were observed at 111-125 amino acid residues (see FIG. 16). However, the peaks for 101-115 peptides were much larger than those for 111-125 peptides. This is considered to be because the larger peak in the 101-115 peptide has a single portion (N-acetylglucosamined portion) according to matrix-assisted laser desorption / ionization mass spectrometry (FIG. 17). -18). From these results, it was suggested that the first O-linked N-acetylglucosamination site of H2B is the 112th serine.

  Then, in order to demonstrate that the 112th serine is a site to be N-acetylglucosamined, a histone H2B mutant was expressed in Escherichia coli and subjected to in vitro OGT assay. In the mutants in which the serine / threonine residues of the histone H2B tail (tail) are each replaced with alanine, the degree of glycosylation is the same except for the 112th serine substitution and the 122th threonine substitution. It did not decrease (see FIGS. 19 to 22).

  However, as described above, in the ETD-MS / MS analysis, the threonine at position 122 is unlikely to be glycosylated, and these two residues of histone H2B are conserved in later animals (see FIG. 23). Because of the structural proximity of the two residues in the α-helix of the H2B protein (see Luger et al., Nature 1997, 389, 6648, page 251), see FIG. It was suggested that this threonine residue is essential for N-acetylglucosamination of the 112th serine (H2B S112 GlcNAc), probably to function as a scaffold for OGT.

Example 4
<Preparation and Evaluation of Antibody that Binds to N-Acetylglucosamined Site of 112th Serine of Histone H2B Protein>
Since the antibody that detects existing O-linked N-acetylglucosamine could not detect glycosylation of endogenous H2B-S112 (data not shown), the H2B 112th serine in living cells. In order to observe the N-acetylglucosamine formation, several antibodies against the H2B peptide in which the 112th serine was N-acetylglucosamined were prepared. From the obtained monoclonal antibody, one capable of specifically detecting N-acetylglucosamine of H2B-S112 with high accuracy was selected and used for the subsequent analysis (hereinafter also referred to as α-H2B-S112-GlcNAc). See Figures 25-27). First, under normal HeLa cell culture conditions, it was confirmed that the 112th serine of H2B was O-linked N-acetylglucosamined to some extent (see FIG. 28). Next, it was revealed that deglucosylation was rapidly caused when the culture medium was a glucose-depleted medium (see FIG. 28). On the other hand, it was revealed that the amount of H2B-S112-GlcNAc recovered when glucose was gradually replenished (see FIG. 29). In particular, it was revealed that the change in the amount of H2B-S112-GlcNAc in response to such a glucose concentration was faster than the cell doubling time of HeLa cells (see FIGS. 29 and 30).

(Example 5)
<Verification of whether or not there is an association between H2BN-acetylglucosamine and H2B monoubiquitination>
Next, since histone modifications interact with each other to determine chromatin status, the effect of H2B-S112-GlcNAc on other histone modifications was examined. That is, from reports on conventional monoubiquitination (“Sun et al., Nature, 2002, 418, 6893, p. 104”, “Minsky et al., Nat Cell Biol, 2008, 10, 10, 4, 483”). In the vicinity of the 112th serine residue of H2B (see FIG. 24), the 120th lysine of H2B was selected for analysis. Data in yeast also suggest that glucose and other carbohydrates cause ubiquitination of H2B 120th lysine (H2B-K120-ub) (Dong et al., J Biol Chem, 2004, 279, No. 3, page 1577). Therefore, in cells cultured for 24 hours under glucose-depleted conditions, glucose treatment was performed to increase the amount of H2B-S112-GlcNAc, and then ubiquitination was observed and analyzed over time. The obtained results are shown in FIGS. As is clear from the results shown in FIGS. 29 and 30, a gradual increase in N-acetylglucosamineation of all proteins and a clear increase in the amount of H2B-S112-GlcNAc detected as RL2 immunoreactive protein occurred after 2 hours. Observed. In addition, a clear up-regulation of the amount of H2B-K120-ub was observed 6 hours after a significant increase in the amount of H2B-S112-GlcNAc.

  Next, the correlation between H2B-S112-GlcNAc and H2B-K120-ub was further examined by an OGT knockdown experiment using a short hairpin RNAi structure against OGT (see FIGS. 31 to 33). The obtained results are shown in FIG. As is clear from the results shown in FIG. 34, in knockdown HeLa cells, the amount of O-linked N-acetylglucosamine in chromatin decreased with a decrease in the amount of H2B-K120-ub (see FIG. 34). Therefore, it was revealed that glycosylation of the 112th serine of H2B promotes monoubiquitination of the 120th lysine of H2B in HeLa cells.

(Example 6)
<Verification of N-acetylglucosamineation of 112th serine of H2B and monoubiquitination of 120th lysine and extracellular glucose and intercellular hexosamine biosynthesis pathway>
In order to verify what kind of sugar metabolic pathway the N-acetylglucosamination of the 112th serine of H2B and the monoubiquitination of the 120th lysine depend on the above-mentioned glucose (Glc) Instead of pyruvate (Pyr) or glucosamine (GlcNAc), the same analysis as described in Example 5 was performed. The obtained results are shown in FIG. As is clear from the results shown in FIG. 35, glucosamine, not pyruvic acid, was able to enhance N-acetylglucosamination of H2B, so that, like other N-acetylglucosamined proteins, the 112th position of H2B. It was suggested that N-acetylglucosamination of serine is dependent on the HBP pathway.

  To further elucidate this point, two inhibitors (DON and AZA) that block the HBP pathway were tested in glycosylation of the 112th serine of H2B caused by glucose. The obtained results are shown in FIG. As is apparent from the results shown in FIG. 35, both inhibitors reduced glycosylation. In particular, the amount of H2B-K120-ub was also down-regulated under conditions where H2B 112th serine glycosylation was reduced.

  Moreover, the same test was done using AA mutation H2B which substituted the amino acid (S112 and T122) essential for N-acetylglucosamination by OGT by alanine. The obtained result is shown in FIG. As is clear from the results shown in FIG. 36, in the overexpression of this mutant, the glucose responsiveness of the H2B-S112-GlcNAc level and the H2B-K120-ub level disappeared. The previous report by the present inventors that N-acetylglucosamine of proteins by OGT depends on the amount of UDP-GlcNAc in cells supplied from extracellular glucose via HBP (see Non-Patent Documents 22 and 23) In addition, these results revealed that the amount of H2B-S112-GlcNAc varies depending on both the extracellular glucose concentration and the state of the HBP pathway, similarly to the amount of H2B-K120-ub.

  Therefore, depending on the increase in extracellular glucose concentration mediated by HBP, the enzyme activity of OGT is enhanced, and then the glycosylation of histone is enhanced, so that the glycosylated histone protein can be used as a detection index of serum glucose level. It became clear that it could be.

(Example 7)
<Analysis of molecular mechanism from N-acetylglucosamination of H2B to ubiquitination of H2B>
The molecular mechanism of H2B-K120-ub that occurs following H2B-S112-GlcNAc found in living cells was analyzed. It has been shown that an N-acetylglucosamine moiety in a saccharide linked to a glycoprotein is provided as a label for E3 ligase-mediated ubiquitination (“Yoshida et al., Nature, 2002, 418, 6896, p. 438), we examined the possibility that H2B ubiquitin E3 ligase is accumulated in N-acetylglucosamined H2B-S112. That is, analysis was performed using the H2B E3 ligase, BRE1A / 1B complex, which has been demonstrated in vitro to bring about monoubiquitination at the 120th lysine site of H2B. The obtained results are shown in FIGS. As is clear from the results shown in FIGS. 37 and 38, co-immunoprecipitation of BRE1A and labeled histone H2B was observed in HeLa cells. On the other hand, the labeled AA mutation H2B did not bind to BRE1A (see FIG. 37). In addition, the binding between endogenous BRE1A and endogenous histone H2B was decreased by glucose depletion (cont.), But rapidly recovered after addition of glucose (Glc) (see FIG. 38).

  Furthermore, in order to confirm these results in vivo, in vitro ubiquitin using N-acetylglucosamined recombinant H2B protein obtained in vitro and recombinant E1, RAD6A (E2), and BRE1A / 1B complex (E3) Chemical analysis was performed. The obtained result is shown in FIG. As is clear from the results shown in FIG. 41, H2B-K120 was monoubiquitinated to some extent by E1 and RAD6A even in the absence of the BRE1 complex. This ubiquitination was significantly promoted in the presence of the E3 BRE1A / 1B complex (see FIGS. 39 and 40). Under these conditions, H2B in which the 112th serine was O-linked N-acetylglucosamined was found to be more efficiently monoubiquitinated (see FIG. 41). Therefore, it was suggested that N-acetylglucosamine of H2B-S112 fixes ubiquitin hololigase that promotes monoubiquitination of H2B-K120.

(Example 8)
<Verification of correlation between authentic chromatin region and H2B S112 GlcNAc site>
Our previous report that OGT is a component of transcriptional co-activation (see Non-Patent Document 23) suggests that H2B-S112-GlcNAc is involved in chromatin activation. So, first of all, PEV using a genetic approach (diversification of the position effect in the group (PEV) in a Drosophila mutant (In (1) W ^m4h ) whose eye color shows a mosaic pattern when the white locus is close to heterochromatin) By the system (see “Zhao et al., Cell, 2008, Vol. 29, No. 1, page 92”), the physiological role of OGT in chromatin reconstitution was verified in OGT mutants. The obtained results are shown in FIGS. As is apparent from the results shown in FIG. 42 and FIG. 43, OGT function-deficient mutant [super sex comb (sxc ¹ )] (“Gambetta et al., Science, 2009, 325, 5936, page 93” When combined with "Sinclair et al., Proc Natl Acad Sci USA, 2009, 106, 32, 13427"), OGT suppresses inactivation of chromatin due to the observed genotype of white eyes It became clear to do.

  Next, in order to more directly evaluate the function of H2B-S112-GlcNAc in the chromatin state, we investigated the localization of chromatin on the fly polytene chromosome using markers that indicate the state of chromatin. The obtained results are shown in FIGS. As apparent from the results shown in FIGS. 44 to 48, the region of H2B-S112-GlcNAc is similar to H2B-K120-ub than the region of H3K9 me2 (the 9th lysine dimethylated from histone H3). Also overlapped with the region of H3K4 me2 (the fourth lysine dimethylated from histone H3) (see FIGS. 44 to 47). The H2B-S112-GlcNAc site was concentrated in the authentic chromatin region in HeLa cells (see FIG. 48). Thus, it was found that H2B-S112-GlcNAc supports chromatin activation rather than chromatin inactivation, at least on the fly polytene chromosome.

  Furthermore, in order to clarify the role of H2B-S112-GlcNAc at the chromosome level, genome-wide location analysis of HeLa cells and analysis of concentrated DNA fragments were performed by chromatin immunoprecipitation using α-H2B-S112-GlcNAc polyclonal antibody. High throughput sequencing was performed. The obtained results are shown in FIGS. As is evident from the results shown in FIGS. 49-52, the H2B-S112-GlcNAc enrichment is widely distributed in the 1088 chromosomal region, half of which are located within 10 kb of the identified Refgenes position. (See FIG. 49). H2B-S112-GlcNAc mainly covered the gene body, but its presence was also confirmed in the promoter and the adjacent downstream region. In the gene expression microarray analysis, transcriptional activity was confirmed in more than half of the identified Refseq genes (see FIG. 50). In addition, the mean profile analysis of the H2B-S112-GlcNAc signal at the metalocus reveals that the H2B-S112-GlcNAc signal is concentrated at the transcription start position (TSS) and maintained up to the transcription end position (TTS). (See FIG. 51). According to Seqpos and TRASFAC motif analysis, it was confirmed that the glycosylation-enriched region contains binding motifs (consensus sequences of transcription factor binding sites) of many transcription factors related to energy metabolism such as HNF4 and COUPTFs ( Table 2, FIG. 52 and FIG. 53).

Example 9
<Verification of the correlation between the transcriptional control in the PGK1 gene and the H2B S112 GlcNAc site>
Next, in order to verify the validity of the genome-wide position analysis described above, the phosphoglycerate kinase 1 (PGK1) gene was selected from the Refseq gene, and histone modification analysis was performed. That is, since the promoter of this gene has an HNF4 binding site and its promoter function is known to be reactive to glucose (data not shown), the modification of histone H2B in the promoter region was examined. . The obtained results are shown in FIG. As is apparent from the results shown in FIG. 54, after high glucose treatment, H2B-S112-GlcNAc increases rapidly within 12 hours, followed by rapid methylation of H2B-K120-ub and H3K4. Increased.

  Therefore, as shown in FIG. 55, depending on the increase in extracellular glucose concentration via HBP, the enzyme activity of OGT is enhanced, and then the glycosylation of histone in the PGK1 gene and the like is enhanced. In addition, it has been clarified that such enhancement of glycosylated histone induces ubiquitination of 120th lysine of histone H2B and methylation of 4th lysine of histone H3, and thus activates transcription of PGK1 gene and the like. It was.

  See also "Folli et al., PLoS One, March 29, 2010, Vol. 5, No. 3, e9923" and "Suhail et al., Indian L Med Res, February 1990, Vol. 92, pp. 21-23"). As described above, the relationship between abnormal expression (enhancement) of PGK1 gene and type 1 (insulin-dependent) diabetes has been clarified, and thus it is considered to be effective against metabolic syndrome such as diabetes using histone protein glycosylation as an index. It is also possible to screen for drug candidate compounds.

  As described above, the present invention makes it possible to detect glycosylation of histone proteins. Furthermore, using the glycosylation of histone proteins as an index, evaluation of serum glucose level, examination of metabolic syndrome, and pharmaceuticals against metabolic syndrome Candidate compounds can be screened. Therefore, the present invention can be used in the medical field such as examination of metabolic syndrome such as diabetes and development of therapeutic drugs in addition to research aimed at elucidating in vivo mechanisms related to glycosylation of histone proteins. is there.

SEQ ID NOs: 1-2
<223> shRNA target sequence SEQ ID NO: 3
<223> O-linked N-acetylglucosamined SEQ ID NOs: 4-15
<223> Sequence of artificially synthesized primer SEQ ID NO: 16-61
<223> Sequence of Artificially Synthesized Polypeptide SEQ ID NO: 67-85
<223> Consensus sequence of transcription factor binding site SEQ ID NOs: 67, 68, 70, 73, 74, 76, 80, 83, and 84
<223> n represents an arbitrary base

Claims (17)

A method for detecting glycosylation of a histone protein comprising the following steps (a) to (b): (a) a step of preparing a sample containing a histone protein (b) a glycosylated histone protein in the sample Detecting. A method for evaluating serum glucose level, comprising detecting a glycosylated histone protein comprising the following steps (a) to (b): (a) preparing a sample containing histone protein (B) detecting a glycosylated histone protein in the sample. A method for examining metabolic syndrome, characterized by detecting a glycosylated histone protein comprising the following steps (a) to (b): (a) a step of preparing a sample containing histone protein ( b) detecting a glycosylated histone protein in the sample. A method for screening a drug candidate compound against metabolic syndrome comprising the following steps (a) to (d): (a) a step of preparing a sample containing histone protein (b) a step of contacting a test compound with the sample (C) detecting the glycosylated histone protein in the sample contacted with the test compound (d) compared to the case where the test compound is not contacted, compared with the case where the test compound is not contacted, of the glycosylated histone protein detected in (c) Selecting a compound that reduces the amount.   The method according to any one of claims 1 to 4, wherein the detection uses an antibody that binds to a glycosylated site of the histone protein.   The method according to any one of claims 1 to 5, wherein the glycosylation is N-acetylglucosamine.   The method according to any one of claims 1 to 6, wherein the histone protein is at least one protein selected from the group consisting of histone H2A and histone H2B.   The histone protein is histone H2B, and the site is at least one serine residue selected from the group consisting of a 91st serine residue, a 112th serine residue, and a 123rd serine residue; The method according to claim 1.   The method according to any one of claims 1 to 8, wherein the glycosylation is N-acetylglucosamination, the histone protein is histone H2B, and the site is the 112th serine residue. .   Antibodies that bind to glycosylated sites of histone proteins   11. The antibody of claim 10, wherein the glycosylation is N-acetylglucosamination.   The antibody according to claim 10 or 11, wherein the histone protein is at least one protein selected from the group consisting of histone H2A and histone H2B.   The histone protein is histone H2B, and the site is at least one serine residue selected from the group consisting of a 91st serine residue, a 112th serine residue, and a 123rd serine residue; The antibody according to any one of claims 10 to 12.   The antibody according to any one of claims 10 to 13, wherein the histone protein is histone H2B, the site is the 112th serine residue, and the glycosylation is N-acetylglucosamination. .   An agent for detecting glycosylation of a histone protein, comprising the antibody according to any one of claims 10 to 14 as an active ingredient.   The chemical | medical agent for evaluating the amount of serum glucose which uses the antibody as described in any one of Claims 10-14 as an active ingredient.   The chemical | medical agent for test | inspecting a metabolic syndrome which uses the antibody as described in any one of Claims 10-14 as an active ingredient.